The long-term goal of our research is to understand how the actin cytoskeleton and endomembrane cooperate in the development and maintenance of healthy photoreceptors. Currently, our grasp of how these dynamic cell functions are integrated in living cells is limited and this in turn limits thinking towards their potential therapeutic manipulation. To remedy this gap, we propose to incorporate live cell imaging and advanced optical methods enabled by fluorescent protein tags and reporters to investigate basic mechanisms of the photoreceptor actin cytoskeleton and endomembrane. A confluence of our recent work on rhodopsin transport and visual arrestin 2 translocation leads us to formulate three specific aims. Building on our observations that fly reticulon, Rtnl1, marks photoreceptor smooth ER, including the subrhabdomeric cisterna essential for photoreceptor maintenance and that Ire1, an initiator of cytoprotective ER signaling, is active and required for normal photoreceptor differentiation, Specific Aim 1 will characterize photoreceptor ER growth and differentiation. It will further test our hypothesis that the ER-associated cytoplasmic isoform of fly Myosin III, NINACp132, contributes a Ca2+-regulated repository of visual arrestin 2 in unstimulated photoreceptors. To test our model of MyoV-mediated rhodopsin transport, Specific Aim 2 will adapt our novel method of light-triggered synchronous release of rhodopsin into the biosynthetic pathway to live cell imaging and use this to assay rhodopsin transport in normal and mutant photoreceptors. FRET will be used to test protein-protein interactions predicted within the MyoV transport complex. We will further examine MyoV phosphorylation control of cargo selection and the role of Ca2+ in MyoV motility. To characterize turnover and regulation of the actin cytoskeleton in living photoreceptors, Specific Aim 3 will track regionally-activated Dendra2 tagged actin within the rhabdomere and terminal web using time lapse imaging. FRET reporters of G-Actin/F-Actin equilibrium and small GTPase activity will be queried during photoreceptor development. Drosophila photoreceptors are an intensively studied model sensory cell utilizing the universal PLC signaling pathway employed by many cell types, including melanopsin-containing ganglion cells of the mammalian retina. If the aims of this project are achieved, key properties of core cell functions will be established in this model.